Method and apparatus for the detection of pathogens, parasites, and toxins

ABSTRACT

The present invention is directed to a method and apparatus for an immunoassay technique that uses amperometric measurements to rapidly analyze different pathogenic microorganisms, including bacteria, viruses, toxins, and parasites. In accordance with one aspect of the present invention, at least one conductive membrane is used to provide support for antibody immobilization and serve as a working electrode; it could also be independent rather than the working electrode. This conductive membrane or powder can be fabricated of a conductive material or can be a nonconductive material over which a conductive material is coated. In either case, the proposed technique is adaptable for use with different materials so as to form a membrane having a pore size that is suited to the particular application. Another aspect of the present invention relates to a compact and simple disposable element that can be easily disposed of after use. In still another aspect of the present invention, the immunoassay can be automated using microprocessor control so as to reduce the amount of human intervention in sample analysis.

BACKGROUND OF THE INVENTION

Pathogens such as bacteria, parasites, toxins, and viruses have emergedas public health problems. Worldwide, pathogenic infections areresponsible for more deaths than any other cause. At times, thepathogens are opportunistic when our resistance is low due to AcquiredImmuno Deficiency Syndrome (AIDS), immunosuppressive drug therapy,anticancer treatment or other related factors. Food borne diseaseoutbreaks, emergence of newer strains of drug-resistant bacterialpathogens without any forewarning (such as the recent outbreak of SevereAcute Respiratory Syndrome (SARS) in Asia), Bird Flu, or pathogens usedas a potentially viable source of biological warfare weapons for massdestruction, necessitate the development of a rapid, portable,analytical device as an early warning system for real-time detection ofbacterial pathogens in field conditions.

U.S. Pat. No. 6,180,335 discloses a flow-through assembly for thedetection of bacterial contamination in the food processing industry.The content of U.S. Pat. No. 6,180,335 is incorporated by referenceherein. The disposable element disclosed in the patent includes animmunosorbent layer having antibodies to a target microbe affixedthereto, a membrane or carbon powder in support of the immunosorbentlayer, and three electrodes for detecting electrochemical signals. Thedisposable element may be used to measure the level of microbiologicalcontamination in a solid sample caused by a predetermined microbe. Thisdisposable sensor element cannot be used as a commercial product.However, the U.S. Pat. No. 6,180,335 is directed to the detection offood contaminations. The disposable element disclosed therein contains apre-filter, three ports, and at least one electrode, all of whichincrease the size of the disposable element and add unnecessary costs tothe assay. Further, in the patent, the filtration membrane used insupport of the immunosorbent layer is not conductive, necessitating thepresence of an electrode in close proximity to the membrane in order toaccurately measure the changes in electron transfer.

The bulky sensor element as shown in FIG. 1 needs continuousreplenishment of Ag/AgCl electrode and chlorinization. Also the carbonworking electrodes must be cleaned. Therefore a disposable element needsto be developed, designed, and tested. For commercialization we need adisposable sensor with Antigen or Antibodies immobilized so any analytescould be readily tested.

SUMMARY OF THE INVENTION

This application describes a rapid, portable, analytical device whichcan be used as an early warning system for real-time detection ofbacterial pathogens in field conditions.

Advantages of the technology described herein include decreased analysistime, increased sensitivity, simplification and automation of themeasuring procedure, which produces quantitative results, decreasedcost, and portability which allows use under non-laboratory and fieldconditions. The technologies provide results in about 20 minutes, arequirement of only five to ten percent of the time of most current testprocedures. A single channel functional prototype device has beenthoroughly tested and used in the experimentation with various analytes.Conceptual designs have been completed for other configurations(including a multi-channel, multi-analyte device) to address a varietyof applications. The portability and speed revealed in the tests willprovide substantial advantages over current practices in these markets.The seriousness of food poisoning alone is exemplified by frequentexposure in the popular press.

The standardized, automated immunosensor diagnostic process, whencombined with the system's reduced size, will permit trainedtechnicians, rather than scientific specialists, to use it onsite aswell as in a laboratory setting. The technology has been developed for anumber of analytes including IgG, IgM, E. Coli O157R7, total E. Coli,Staphylococcus sp and Hanta-virus. Tests for Salmonella and Hepatitisare in process.

The technology provides a general methodology for fast, sensitive,inexpensive and portable immunoassays over a wide range of analytes suchas bacteria, viruses and chemicals. Since this technology offers afaster and cheaper method of employing test procedures that are alreadyapproved, regulatory issues are dramatically reduced. Additionally,current conventional immunoassay techniques are comparatively lengthyanalyses that usually take several hours.

The immunosensor improves upon conventional immunoassay techniques bythe enhancement of immunointeraction efficiency; this is accomplished byusing a flow-injection assay technique, which employs immunocolumns.This provides a high area-to-volume ratio of solid-to-liquid phase andleads to a high rate of immunointeraction due to reduced diffusionlimitations. Another area of improvement is the development of a fasterand more sensitive detection method using electrochemical detection ofthe labeled immunospecies.

The potential applications of this novel immunoassay are based upon itsadvantages relative to existing techniques, namely:

-   -   1) It is 15 to 20 times faster    -   2) It is a highly-automated assay that can be conducted by        less-trained personnel    -   3) It can be configured as a portable device, which allows        assays to be conducted in the field.

The technology is based on a “sandwich” scheme, which is more sensitivethan the usual “displacement” scheme. It is especially effective forlarge molecular-weight analytes, which represent the vast majority ofapplications in the target industries.

The present invention is directed to an immunoassay device and adisposable sensing element that uses amperometric measurements torapidly detect and analyze different pathogenic microorganisms,including bacteria and viruses, toxins and parasites. In accordance withone aspect of the present invention, at least one conductive membraneand/or the immunosorbent powder is used to provide support for antibodyimmobilization and serve as a working electrode, or it could be used byitself without being a working electrode. This conductive membrane canbe fabricated of a conductive material or can be a nonconductivematerial over which a conductive material is coated.

In either case, the proposed technique is adaptable for use withdifferent materials such as carbon powder or so as to form a membranehaving a pore size that is suited to the particular application. Anotheraspect of the present invention relates to a compact and simpledisposable element that can be easily assembled and disassembled. Instill another aspect of the present invention, the immunoassay is nowautomated using microprocessor control so as to reduce the amount ofhuman intervention in sample analysis.

In summary, this broad-based technology was developed using a“sandwich-scheme” immunocolumn with enzymatic labeling and amperometricsignals. It has been incorporated in a semi automated, portable,functional prototype which provides for highly-sensitive, quantitativedetection of levels of most large molecules in a sample. In addition todetecting pathogenic infections, applications are numerous in suchfields as medical and veterinary diagnostics, food processing qualitycontrol, epidemiology field analysis, and environmental chemicalanalyses. Anticipated products include a variety of automated deviceswith substantial competitive advantages and a series of disposableflow-through immunocolumns specific to each analyte to be diagnosed.Proprietary cartridges of commercially available immunochemicals will bepart of the automated process.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic of the flow through immunosensor assembly (1)immunocolumn (2) filtration membrane (3) layer of carbon deposited ontop of the membrane (4) counter electrode (5) reference electrode (6)glass capillary and (7) current collectors [13].

FIG. 2 is Cyclic voltammograms of a spin-coated nylon membrane.

FIG. 2( a) is a Cyclic Voltammograms of a dip-coated nylon membrane.Conditions: scan rate of 20 mv/sec; iodine concentration of 1×10⁻⁴, 20mM phosphate buffer (pH 5.

FIG. 2( b) is a Cyclic Voltammograms of a spin-coated nylon membrane.Conditions: scan rate of 20 mv/sec; iodine concentration of 1×10⁻⁴, 20mM phosphate buffer (pH 5.8).

FIG. 3 (a) Calibration curves for amprometric detection of total E.coli. The lowest detectable concentration is 50 cells/ml at a flow rateof 100 μl/min. All measurements were done in triplicate (n=3) and datapoints are presented as mean±S.D. (b) Calibration curves for amprometricdetection of L. monocytogenes. The lowest detection concentration is 10cells/ml at a flow rate of 100 μl/min. All measurements were done intriplicate (n=3) and data points are presented as mean±S.D. (c)Calibration curves for amprometric detection of C. Jejuni. The lowestdetection concentration is 50 cells/ml at a flow rate of 100 μl/min. Allmeasurements were done in triplicate (n=3) and data points are presentedas mean±S.D. Background is indicated by dashed line

FIG. 4 shows the effect of graphite layer thickness on the amperometricsignal resulting from iodine reduction. Thus, in a preferred embodiment,membranes thickness is about 450 μm.

FIG. 5 is a representation of immunofilteration membrane made from Toraycarbon paper with determined by an Atomic Force Microscope.

FIG. 6 a is an SEM microphotograph of the carbon paper.

FIG. 6 b contains physical data for a variety of different paperthicknesses, provided by the manufacturer.

FIG. 7 is a photo of portable automated immunosensor device.

FIG. 8 shows the calibration curve for E. coli using optimizedimmunoassay procedure showing linear dependence on concentration overworking range (50-1000 cells/ml).

FIG. 9 shows a screen-printed commercially available disposable sensorelement.

FIG. 10 is the design of the flow-injection cell for the disposablesensing element: 1—flow cell with in and out flow, 2—Carbon counterelectrode, 3—Carbon working electrode, 4—Ag/AgCl reference electrode.

FIG. 11 shows the design of the new multi channel disposable sensingelement: 1—flow cell with in and out flow, 2—Carbon counter electrode,3—Carbon working electrode, 4—Ag/AgCl reference electrode,5—five-channel injection micro valve manifold.

FIG. 12 Schematic layout of the immunosensor device

FIG. 13 Wiring Diagram for Potentiostat

FIG. 14 Wiring diagram for the pumping system. The pumping systemconsists of an Instech Model P625 Peristaltic pump and HEX NC ManifoldValve Model 225T09 (Neptune Research and Development Inc., New Jersey,USA). This Manifold Valve system incorporates six separatednormally-closed Teflon isolation valves integral to a single block ofTeflon. The six valves had independent inlets and one common outlet. Thesignals that controlled these devices are 5v digital signals, but thepump was a 9v device and the valves were 12v devices. So the system usedMOSFETS to drive the devices from the 5v signals.

FIG. 15 Table 1 shows the response of the system using the twoconjugates peroxidase labeled and alkaline phosphatase labeledanti-rabbit IgG.

FIG. 15 Table 2: Shows the response for Rabbit IgG as analyte andperoxidase labeled anti-rabbit IgG as the conjugate.

FIG. 15 Table 3 Deer mice blind blood samples and CDC data were given usform Dr. Terry Yates (Museum of Southwestern Biology, Department ofBiology, University of New Mexico).

FIG. 16 Current response for various potentials for (A) a standardpotentistat, (B)Potentiostat circuit in a buffer solution.

FIG. 17 Wiring Diagram for the Microcontroller.

DETAILED DESCRIPTION OF THE INVENTION

One aspect of the present invention relates to the use of conductivepowder or membrane in an immunoassay, which acts both as a solid phasesupport for antibody immobilization and or also as a working electrode.Since the immuno-interactions and amperometric measurement occur at thesame sites, a significant increase in the magnitude of theelectrochemical response is resulted.

In one embodiment, non-conductive membrane such nylon membrane is coatedwith a conductive material, such as graphite powder. Graphite can bedeposited onto membranes in different ways, including, but not limitedto, dip coating, spin coating, and vapor coating. In operation, graphitepower is first mixed with a suitable solution to form slurry. In onepreferred embodiment, cellulose acetate dissolved in acetone is used.Subsequently, a piece of nylon membrane can be dipped into the slurryfor coating. As an alternative a spin coater, such as the one with modelno. EC101 manufactured by Headway research Inc, Garland, Tex., can beused to coat the membrane. The coated membranes are then dried at roomtemperature before use.

Membranes prepared by different coating methods may possess differentcharacteristics. For example, the spin-coating technique produced a wellcontrolled, thin, and evenly distributed graphite layer, as shown by theSEM photographs. On the other hand, graphite layers formed bydip-coating technique were less controlled and thicker. See FIG. 2.Further, when using the same nylon membrane and graphite slurry, thepores size produced by spin coating is smaller than that produced bydip-coating.

However, dip-coated membranes exhibit better electrochemical propertieswhen compared with spin-coated membranes, as shown in FIGS. 2 a and 2 bby cyclic voltammetry. Specifically, iodine reduction peaks were morepronounced in the dip-coated membranes (FIG. 3). Furthermore, thedifference in current magnitude between the CV in absence and presenceof iodine was much greater in the case of dip-coated membranes. This maybe attributed to the higher surface areas of graphite that is available,as shown from the SEM photographs.

The thickness of the graphite film formed on the nylon membrane can becontrolled by how many times the membrane is dipped into the slurry or,in the case of a spin coating, the speed and duration of the spin. Ithas been observed that increased thickness in a certain range may causean increase in amperometric signal due to the increased electro-activearea. However, further increase in graphite layer thickness may resultin a decreased amperometric signal because of the appearance of cracksin the graphite layer, which in turn may cause electrical discontinuityand hence a smaller surface area as shown in FIG. 4.

The choice of a suitable membrane pore sizes is crucial for achievinghigh sensitivity. This is because membranes with too small pores maycause the deposition of carbon/graphite might cause partial blockage ofthe membrane pores, and consequently a non-specific, physical entrapmentof pathogen as well as increased flow resistance. On the other hand,membrane with too big pores may result in a great portion of theimmunoassay solution to flow through the membrane without having enoughcontacts of the immunosorbents. Consequently, nylon membranes withdifferent pore sizes have been tested and it appears that nylon membranehaving a pore-size of 5 μm provides an optimal result for graphitedeposition, allowing the formation of a conductive layer while stillmaintaining adequate flow properties.

The solution used to mix with graphite powder to form slurry may alsoaffect the electrochemical properties of the membrane. Specifically, ithas been observed that the ratio of cellulose acetate to acetone isimportant. As shown in FIG. 6, the amperometric signal is low for bothhigh and low concentrations of cellulose acetate. This might be becausethat a high concentration of cellulose acetate (65% v/v in acetone)results in a highly viscous membrane that clumps and cracks upon dryingwhile a very low concentrations of cellulose acetate (35% v/v inacetone) results in a highly fluid composition that is difficult to castevenly over the nylon membrane. Thus, as a preferred embodiment, acellulose acetate concentration of 50% v/v in acetone can be used toproduce a membrane with acceptable amperometric response and ease ofcasting.

The graphite-coated membranes can be further processed for immobilizingthe desired antigen or antibody by well-established method in the art,such as the use of Woodward's Reagent K. The present invention shouldnot be limited to the one embodiment wherein nylon membranes were coatedwith graphite powders. Other types of porous membranes recognized in theart, such as carbon fiber paper, polycarbonate membrane, etc., may alsobe used as the underlying membrane for coating. The coating materialused in the present invention can be any conductive material that iscapable of being deposited on porous membranes, including, but notlimited to, graphite/carbon, conductive polymer, etc. Further, thesubjects that can be immobilized on the resulted conductive membrane canbe either antibodies or antigens. Even further, the conductive membranecan be used in a reaction chamber as shown in FIG. 7 or in aflow-through immunoassay device as disclosed previously or in thepresent invention below.

Another aspect of the current invention relates to the use of aconductive membrane, such as the carbon paper manufactured by TorayIndustries, Inc., Tokyo, Japan, directly in an immunoassay withoutcoating. Because of the immunosorbent and conductive nature of theconductive membrane, it served both as a support for immobilizingimmunoagents and, at the same time, as an electrode for electrochemicalmeasuring.

A design based on the use of Toray carbon paper as the solid support forthe immunofiltration membrane is shown in FIG. 5. In such an instance,the carbon paper offers a large surface area to volume ratio and, at thesame time, it could function as an electrode. The carbon paper consistsof pores whose size is on the same order of magnitude as some targetanalytes, making it ideal for filtration. The carbon itself possessescharacteristic pores whose size is of the same order as most antibodies,making it also ideal as an immunosorbent. However, there are times whenthe carbon paper is not completely satisfactory with respect to its poresize or other characteristics. In such cases, the carbon paper can becoated by the coating method disclosed in the current invention. An SEMmicrophotograph of the carbon paper is shown in FIG. 6 a.

The use of conductive membrane such as carbon paper or graphite coatednylon membrane as an immunofiltration membrane has many advantages. Forexample, the conductive membrane has high tensile strength and highmodulus, good handling and flexibility, excellent thickness uniformity,and minimal electrochemical corrosion. Further, the use of conductivemembrane eliminates the problems associated with construction of acomplex disposable sensing element used in previous heterogeneous flowimmunosensors. Disposable sensing elements have previously consisted ofplastic columns with membrane filters on which the immunosorbent isdeposited by centrifugation in order to form an immunoelectrode to beused in electrochemical detection. This requires extensive preparationin order to create, driving up the cost of the assay and increasingpossibilities for error. However, using carbon paper or other coatedmembranes as an immunofiltration membrane as well as an immunoelectrodewould simplify the system, reduce the cost, and meanwhile increase thesensitivity and reliability of the system.

Further, the traditional heterogeneous immunosensor requires theregeneration of the immunosorbent after each assay, a process involvesthe use of chaiotropic reagents to break the antigen-antibody bond andinevitably results in a loss of enzyme activity increases the cost andcomplexity of the assay. The use of a conductive immunofiltrationmembrane, either coated or not coated, permits the construction ofimmunoassay elements that are disposable and inexpensive. This wouldsimplify the assay procedure and reduce its cost and at the same timeensure constant activity of the immobilized immunoagents without theneed for regeneration of the support.

Another aspect of the present invention relates to a compact yetlow-cost disposable unit for flow-through immunoassay.

The focus on development of a disposable sensing element for anautomated simple single or more channels on-line flow-injectionimmunoassay amplification procedure and amperometric biosensor prototypethat we have already developed for fast, quantitative detection of lowconcentrations of total E. coli, S. Aureus hantavirus and Influenzas.Qualitative detection of low concentrations of Hantavirus, Influenza A,Bird Flu, and Para influenza. The system will be arranged as a portabledevice. The photo of the device is shown in FIG. 7, all components areeasily accessible allowing ease of service and maintenance. Furtherutilization of amperometric detection in comparison to spectrophotometerallows for the substantial limitation of reagent consumption, eliminatesoptical elements from the measuring device. And the use of higherworking dilutions of the sample (1:200-1:500) minimizes the interferenceeffects and allows one to ignore the difference in the color andturbidity of natural samples for hanta virus. Our device was found to berobust in field operations: in terms of accuracy, reproducibility, andease of use, sensitivity and ability to compensate for interference. Thedevice exhibits good response stability. The sensitivity of theimmunosensor in this preliminary evaluation had no false positive orfalse negative samples with 100% accuracy using hanta virus rodentblood.

Testing the new circuit with optimization using the old sensor system,the overall assay time was found to be 17 min. This optimized assaycould detect the target analyte 50-100 cells/ml, in a linear fashion asshown in FIG. 8. Higher concentration of analyte were analyzed, however,the results were not linear. This indicates that there is a saturationpoint in the system that is slightly higher than the maximum value ofthe described working range. To evaluate the immunosensor performance inreal samples for use in medical applications, samples of dialysate frompatients were evaluated to determine if E. coli was present, which couldlead to peritonitis. First, sterile dialysate was spiked with knownconcentrations of bacteria to generate a calibration curve for thesensor, as well as the ELISA, which is shown below in FIG. 8. Next,blind samples that were known to have bacterial contamination wereassayed using the newly developed sensor, with the old sensing elementand the results were compared to that of a standard ELISA, shown in FIG.8. It can be seen that the sensor system gives results that are withinfew percentage points of the standard ELISA values. However, theimmunosensor quantified the bacteria in 17 minutes, while preparationand use of the ELISA took two full days. Also, a much smaller volume ofimmunochemicals can be utilized by the sensor, which can lead tosignificant cost decrease if many samples are being tested. This meansthat the sensor can be a good alternative to the ELISA, and has thepotential to be used in several similar medical applications.

Engineering of the Disposable Sensing Element

A disposable sensing elements design, development and standardization ofThe disposable sensing elements will be engineered in the form of a oneor more channel cartridge as illustrated in FIG. 9 for simultaneousmeasurement of total E. coli, and S Aureus, hanta virus and bird Flu inbiosamples without pre-enrichment.

The setup with a printed screen disposable element could function withthe electric circuit described in FIG. 8. The device should exhibit goodresponse stability when tested earlier with our bulky sensor element.The sensitivity of the immunosensor was 100% in this preliminaryevaluation, with no false positive or false negative samples using hantavirus rodent blood. We will use a special filter paper and immobilizethe antibodies on it. Also we have to build in this proposal a flow cellmade out of glass or plastic and fixed over the commerciallyscreen-printed electrode, which is shown in FIG. 10.

The special filter paper with covalent immobilized antibodies will beattached onto the working electrode site of screen-printed electrode,and will play both of roles immunosorbent and working electrode withextremely high porosity and high surface area. Our previous resultsusing this special filter paper or carbon particles as immunosorbent andworking electrode simultaneously allows delectation of bacteria withhigh sensitivity and selectivity. The inner volume of the cell is about55 μL. This design of disposable sensing element offers an easy andconvenient way to use and commercialize the sensor.

Engineering of the Flow-Injection Enzyme Immunoassay Procedure with theDisposable Sensing Elements

Testing of different concentration of Hantavirus, Bird Flu, E. Coli andS Aureus modifying the design to confirm our ELISA data. Initial studieswill use laboratory cultures of coliform bacteria at known low and highcell concentration. The total number of bacteria in each sample will beknown.

Engineering Design of the on-Line Flow-Injection Enzyme ImmunoassayProcedure

The main characteristics of the portable flow-injection immunoassayprocedure (flow rate, incubation time, concentrations of conjugate andsubstrates, volume of sample, working temperature, configuration andgeometric parameters of the flow-through electrochemical sensor) will beinvestigated in order to increase the sensitivity of the assay, anddecrease the time of measurement. The optimal antibodies forimmunosensors will be chosen on the basis of measured kinetic constants.FIG. 11 describes the laser engraved channels for the flow of analytesconnected to the electronic circuit in FIG. 17 with electronic valves.The disposable element can be designed and manufactured at the Center ofHigh Technology Materials at UNM (CHTM). The flow channels have to becovered with either glass or plastic material. The effect of suchfactors as the pore size and thickness of membrane, flow rates andtemperature on the transport phenomena will be also investigated.

The main characteristics of the portable flow-injection immunoassayprocedure (flow rate, incubation time, concentrations of conjugate andsubstrates, volume of sample, working temperature, configuration andgeometric parameters of the flow-through electrochemical sensor) will beinvestigated in order to increase the sensitivity of the assay, anddecrease the time of measurement. The optimal antibodies forimmunosensors will be chosen on the basis of measured kinetic constants.FIG. 11 describes the laser engraved channels for the flow of analytesconnected to the electronic circuit in FIG. 5 with electronic valves.The disposable element can be designed and manufactured at the Center ofHigh Technology Materials at UNM (CHTM). The flow channels have to becovered with either glass or plastic material. The effect of suchfactors as the pore size and thickness of membrane, flow rates andtemperature on the transport phenomena will be also investigated.

Fabrication of One or More Electrochemical Biosensor Prototype

Development of an automated, one or more channel electrochemicalbiosensor prototype will be used to develop one channel, automatedelectrochemical biosensor. Additional Channels can be added to thesystem consists of more channels amperometric disposable sensing elementin a rotating cartridge, will be used.

Preferably, the disposable unit as illustrated in FIG. 11 (disposableelement) other shapes may also be used. The disposable unit ispreferably made of plastic or glass or other non-conductive materials.The cylindrical or any other shape column is hollow, having an innercapacity surrounded by the cylindrical walls. The cylindrical column orany other shape column further comprises two ends, an upper end and alower end, with openings at each end. The upper opening is connected tothe inlet of different solutions, thus opens the inner capacity of thecylindrical column or any other shape to accept the insertion of anobject or tube connection that has a smaller diameter. On the otherhand, the solution will fall onto the membrane or filter affixed on thecenter carbon electrode's (the working electrode) lower opening lettingthe flow exit the device after it passes through or over the membrane orthe particles and be collected as shown in FIG. 10.

In one embodiment of the present invention, the membrane is placed onthe Carbon electrode that is a screen printed or micro fluidicsfabricated. This carbon membrane is conductive by itself, such as thecarbon paper manufactured by Toray Industries, Inc., Tokyo, Japan. Itcan be either coated or not coated by a conductive material as describedabove in the present invention. In another embodiment, the membraneitself is nonconductive, but is coated with a conductive material suchas graphite so that the resulted membrane is conductive. In bothembodiments, the membranes are capable of being a support forimmobilizing immunoreagents and, at the same time, an electrode.

In yet another embodiment, highly dispersed fine particles of conductivematerial such as carbon can be used. In such an instance, the membranefunctions primarily as a supporting material to prevent the carbonparticles from falling through the lower opening of the column. It isimmaterial whether the membrane is conductive or not. In a preferredembodiment, highly dispersed carbon particles (ULTI) are used.

A preferred embodiment: The entire assembling process in the previoussensor has been eliminated and now it is simpler and easy to operate. Sois the disassembling process after the completion of an experiment. Forevery new immunoassay, a new disposable unit is used, ensuring bothaccuracy and efficiency. Further, an array of the disposable unit can beprepared, having different antibodies or antigens immobilized on thesurface of the immunosorbents for the detection of a wide range ofpathogens, including, but not limited to, bacteria, viruses, parasites,or toxins.

The present invention further relates to an automated system offlow-through immunoassay. In a preferred embodiment, the automatedsystem consists of four blocks: a power supply, a potentiostat, apumping system, and a microcontroller. The power supply uses eitherbatteries or 110 AC to generate all the voltages used in the device. Thepotentiostat is used to bias the immuno-electrode and convert itscurrent into a voltage signal. The pumping system delivers the reagentsused to test the sample. And the microcontroller runs the program usedto test the sample and display the results. FIG. 12 shows the schematicof the immunosensor layout.

During operation, the microcontroller switches the pump on and thefluids are pumped through the manifold valve system. The microcontrolleris programmed so that it opens a particular valve during a particularstage of the assay, and a peristaltic pump pumps the required fluidthrough the valve. The pump speed is adjusted by adjusting the speedrange pot so that a required flow rate is obtained.

The automated immunoassay system may also comprise two push-buttonswitches and a liquid crystal display module (LCD). First button is the“start/yes” button and the second is the “no” button. When all therequired fluids are filled and the electrodes are connected to thepotentiostat, the assay procedure is started by pushing the “start/yes”button. The LCD displays the stages of the assay procedure. When theassay procedure is in the measuring stage, voltage is applied betweenthe working and counter electrodes by potentiostat. The timing ofapplication of the potential is controlled by the microcontroller. Whenthe assay procedure is finished, the LCD displays the measurements.

Preferably, the performance of the system is evaluated before use. Thefirst step of evaluation is to test the flow rate of the fluids throughthe valves. The speed range pot on the peristaltic pump is adjusted toadjust the pumping speed. The flow rate is measured by collecting fluidfrom the valves for a known time. The speed range pot is adjusted tillthe desired flow rate is obtained.

The next step is to evaluate the potentiostat circuit by connecting itto a buffer solution and applying a potential. Varying the resistancevaries the potential applied and the output current is measured.Similarly a standard potentiostat is taken and various potentials areapplied and the output current is noted. A graph between current andvoltage is plotted. Both the results gave almost a linear dependence(FIG. 13).

Further, the resistance is adjusted so that the desired potential isapplied by the potentiostat circuit. Then buffer was pumped through thevalves and the buffer flowing from the valves was collected for eachstage. The collected buffer was measured and the flow time of each stagewas adjusted by programming the microcontroller, so that the requiredamount of fluid was flowing in each stage.

There are many advantages by using the automated immunoassay system asdisclosed in the current invention. The assay is completely automated sothat little human intervention is needed to complete an assay. Further,the assay can be stopped or started at any stage of the process. Thetotal assay time is reduced when compared with the time required formanual assaying and the data acquisition can be connected to a computeror data storage device for analysis or storing. Additionally, differentassay procedures can be programmed into the microcontroller so that thesystem can be easily adapted for the detection of different pathogens.

The current invention can be better illustrated by the followingexamples.

Example 1 Coating Nylon Membrane with Graphite Powder

Nylon membranes were obtained From Pall Corporation, New York, N.Y.(Biodyn™ B, C, with pore sizes of 0.45, 1.2, 3.0, and 5.0 μm.) Graphitepowder was obtained from Fischer Scientific, Pittsburgh, Pa., which wassieved by 53 μm mesh to obtain a particle size of 53 μm or lower.

When using the dip coating method, 1.0 g of the sieved graphite powderwas mixed in 1.0 ml of cellulose acetate solutions of 25%, 50% and 75%(v/v) in acetone to form a slurry and a piece of nylon membrane (2×4 cm)was dipped in the slurry for a number of times to form layers ofgraphite coating. The dip-coated membranes were then dried at roomtemperature before use.

When coating the nylon membrane, a spin coater model no. EC101,manufactured by Headway research Inc., Garland, Tex., was used. The sameabove mentioned slurry was used to coat the nylon membranes. The speedof 4000 rpm was used for different amount of time (e.g. 30, 60, 90 and120 seconds) to achieve layers with different thickness.

Membranes prepared using the spin-coating and the dip-coating methodswere tested to verify conductivity and other characteristics. Theresistance of graphite-coated nylon membranes prepared by both methodswas about 0.38Ω. Further, as shown by the SEM photographs, thespin-coating technique produced a well controlled, thin, and evenlydistributed graphite layer. The pores size produced by spin-coating wasabout 0.5 μm. On the other hand, graphite layers formed by dip-coatingtechnique were less controlled and thicker, with the average pore-sizeof about 3 μm, which was much higher than the one produced byspin-coating.

The electrochemical properties of the graphite coated nylon membraneswere further evaluated by cyclic voltammetry. As shown in FIGS. 2 a and2 b, iodine reduction peaks were more pronounced in the dip-coatedmembranes. Furthermore, the difference in current magnitude between theCV in absence and presence of iodine was much greater in the case ofdip-coated membranes. This may be attributed to the higher surface areasof graphite that is available, as shown from the SEM photographs.

The graphite coated membranes were further processed for immobilizingthe desired antigen or antibody. In one embodiment of the invention, E.Coli antibodies were immobilized on the graphite coated membranes.Specifically, the membranes were first cut into one centimeter squaresand placed into separate wells of a polystyrene plate. The membraneswere then immersed in 1 ml of 20 mg/ml solution of Woodward's Reagent K(pH 4.5) and incubated with simultaneous shaking for 2 hours at roomtemperature. The membranes were washed three times (3 minutes each time)in 1 ml of 20 mM Na-phosphate buffer solution (pH 7.8). The membraneswere transferred to a new polystyrene plate and left for 20 minutes todry. Consequently, 20 μl stock solution (3 mg/ml) of anti-E. Coliantibodies were dropped onto each membrane and left at room temperaturefor 20 minutes to dry. The membrane was then incubated for 2 hours with5 mg/ml of trypsin inhibitor prepared in 0.1 M phosphate buffer (pH 7.8)and stored at 4° C. until further use.

Different concentrations of E. Coli cells were added to each antibodymodified membrane and incubated at 20° C. for ten minutes. The membraneswere washed 3 times (3 minutes each) in 1 ml of 20 mM phosphate buffersolution (pH 7.8) to remove any unbounded E. Coli cells. Then 10 μg/mlsolution of anti-E. Coli horseradish peroxidase (HRP) conjugate wereadded to each membrane and again incubated at 20° C. for ten minutes.The membranes were washed 3 times (3 minutes each) in PBST (pH 5.6).Each membrane was then immersed in the substrate solution (0.1 mMNaI+0.1 mM H₂O₂) to measure the electrochemical current at +0.105V. Theset up used for the amperometric measurements is shown above.

As shown in the equations below, HRP catalyzes the oxidation of iodideinto iodine. Electrochemical reduction of iodine forms the basis ofdetermination of the activity of HRP enzyme and quantification of theenzyme label.NaI+H2O2-----HRP--->I22e−+I2-------Electrode----->2I-

FIG. 3 shows a calibration curve obtained for amperometric measurementof iodine using the optimized dip-coated membrane. It can be seen thatconcentrations as low as 0.1 mM of iodine were detected, demonstratingthat the membrane has excellent sensitivity and superior electrochemicalperformance.

Application of the optimized membranes to the detection of E. Coli, L.monocytogenes and campylobacter C. Jejuni concentration is 40 cells/ml,is shown in FIG. 14.

It was observed that the lower detection limit of total E. Coli, of L.monocytogenes, and campylobacter C. Jejuni concentration is 40 cells/ml,with a readily detectable range form 40 to 1000 cells/ml. When the E.Coli concentration was greater than 1000 cells/ml, a decreasedamperometric measurement was obtained, indicating a “hook effect”wherein the electrode surface was partially blocked by E. Coli bacteria.

Example 2 The Detection of Hantivirus in Mice Blood Materials

Rabbit IgG, anti rabbit-IgG antibodies, peroxidase labeled Goat anti-IgG(conjugate), peroxidase labeled Goat anti-peromyscus leucopus IgG,phosphatase labeled Goat anti-peromyscus leucopus IgG were from SigmaChemical Co. (MO, USA). Recombinant replica of Sin Nombre Virus (SNV)protein was obtained from Dr. Brian Hjelle, Department of Pathology,UNM, in kind support. Sodium phosphate (Monobasic), sodium phosphate(Dibasic), sodium acetate (Trihydrate), Sulphuric Acid were from FisherScientific Co. (NJ, USA). Sodium chloride, sodium hydroxide were from JTBaker. (NJ, USA). Tween 20 (Polyxyethylenesorbitan Monolaurate) was fromSigma Chemical Co. (MO, USA). ULTI (Ultra Low Temperature IsotropicCarbon) was from Carbonmedics Inc. (TX, USA). Woodward's reagent K(N-ethyl-5-phenylisoxazolium-3′-sulfonate), Trypsin inhibitor, BovineSerum Albumin, Hydrogen Peroxide (30% v/v aqueous solution), α-Naphthylphosphate were from Sigma Chemical Co. (MO, USA). Hydrochloric acid,Sodium Hydroxide, Sodium Iodide were from JT Baker (NJ, USA).

Sensor Design

The immunoassay system design concept is based on conductingenzyme-linked assays by utilizing flow of analyte and immunochemicalsthrough porous immunosorbents (carbon) coupled with electrochemicalbased transduction mechanisms as a means of overcoming the limitationassociated with conventional techniques. From the engineeringperspective, the use of flow-based systems allows for easier automationof the analysis procedure since pumps usually drive the flow and valvesthat are easily controlled by microprocessor based technology.

Preparation of Immunosorbent

Woodward's reagent K immobilization is a technique for obtainingcovalent linkage of the proteins to the surface of the carbon(covalently linked immunoreagent-solid phase conjugates). First, anactivation of the solid support is performed. Second, coupling of theantibody to the activated solid support occurs. It does not leave tracesof itself after the process.

The pH of the solution with Woodward's reagent K (20 mg/ml) in water wasadjusted to 4.5 using diluted NaOH solution, followed by suspension of25 mg of Ultra Low Temperature Isotropic Carbon (Product of CarbonmedicsInc.) in 1 mL of it. This is followed by incubation at room temperaturefor 2 hours with shaking. The suspension was later washed 5 times withdistilled water by repeated centrifugation and removal of thesupernatant. Carbon thus treated with Woodward's reagent K was suspendedin 1 mL of a solution of IgG (0.5 mg/mL). The suspension was incubatedat room temperature for 2 hours with shaking. After incubation, thecarbon particles are again washed for five times with distilled waterwith repeated centrifugation and removal of the supernatant. 5 mg oftrypsin inhibitor is then added to the same suspension as a blockingagent and incubated for an additional 2 hours at room temperature withshaking. The suspension was finally washed 5 times with PBS by repeatedcentrifugation (5 minutes each) and removal of the supernatant. Theimmuno-sorbent was stored in the same buffer solution at 4° C. Theimmobilization of recombinant nucleocapsid hantavirus protein antibodies(10 μg/mL) is performed similarly.

The System

The power supply system used DC-to-DC converters to generate the +12 V,+9 V, +5 V, and −5 V signals needed by the other systems. A pair of 9 Vbatteries was used for portable power, but a relay will disconnect thebatteries if a 12 V power pack was plugged into the external jack.

An AT89C51, 8-bit microcontroller was used, which was a low-power,high-performance CMOS microcomputer with 4K bytes flash programmable anderasable read only memory (PEROM). The microcontroller was the heart ofthe system and controlled the functioning of all the electronic parts inthe system. The micro controller system used an 8051 chip to run thetest program for the device. It generated the control signals for thepumping system, used an analog to digital converter to acquire data fromthe potentiostat, and displayed the results on an LCD display.

The potentiostat used a constant current source to supply the biasing ofthe immuno-electrode, and instrumentation amplifiers to convert theelectrode current into a voltage signal. The system also used digitalswitches to ground the electrode when it isn't being used. The requiredpotential between the working and the counter electrode was applied andthe output signal i.e., the current between the working and thereference electrode was measured by the potentiostat.

The pumping system consists of an Instech Model P625 Peristaltic pumpand HEX NC Manifold Valve Model 225T09 (Neptune Research and DevelopmentInc., New Jersey, USA). This Manifold Valve system incorporates sixseparated normally-closed Teflon isolation valves integral to a singleblock of Teflon. The six valves had independent inlets and one commonoutlet. The signals that controlled these devices are 5 v digitalsignals, but the pump was a 9 v device and the valves were 12 v devices.So the system used MOSFETS to drive the devices from the 5 v signals.

The hantavirus assay had six stages of pumping. The first stage, themicrocontroller was programmed to pump 0.02 M Na-phosphate buffer (pH7.8) containing 0.15 M NaCl and 0.1% Tween-20 (PBST) for 1 minutethrough valve 1. This was a pre-washing stage, wherein the immunosorbantwas washed with the PBST. The second stage was sample injection stagewhere the microcontroller pumped PBST containing blood samples, testedfor Hantavirus IgG antibodies (target analyte) through valve 2, for 3min. In this stage the target analyte was bound to the surface of theimmunoelectrode. Stage three was washing stage where the microcontrollerwas programmed to open valve one and pump PBST for 2 minutes. In stagefour the conjugate was injected. The microcontroller opened valve 3 andpumped 2 ug/ml of peroxidase-labeled anti-human IgG (conjugate) againstthe target analyte flow through the immunocolumn for 8.5 min. Stage fivewas again a washing stage where 0.05 M Na-bicarbonate buffer solution(pH 9.6), containing 0.01 M NaOH and 0.15 M NaCl was pumped throughvalve 4 for 4 min by the microcontroller. In the final stage, themicrocontroller pumped 0.05 M Na-bicarbonate buffer solution (pH 9.6),containing 0.01 M NaOH and 0.15 M NaCl, 0.1 mM α-Naphthyl phosphate,flow through the valve 5 for 5 min. The amperometric measurements wereperformed at a fixed electrode potential (+105 mV vs. Ag/AgCl) which wasapplied by the potentiostat.

Methodology

Highly dispersed carbon or carbon filter paper offers a large surfacearea and at the same time functions as the working electrode because ofits conductive nature or it can work without being the electrode. Thematerial possesses characteristic area per unit mass and the particlesizes lie in the same order of magnitude as that of proteins. Thisprovides the basis for immobilization of antibodies on this carbon. Sucha process enhances the proximity of biological components with thetransducer, a very essential factor for biosensor development. ULTI isused as a material for the working electrode in a finely dispersedpowder form (fraction less than 275 mesh size of an ASTM sieve). Currentcollection is achieved through a carbon rod, while the reference Ag/AgCland carbon represent the reference electrode and the counter electroderespectively. Amperometry is a technique where the output of the sensoris current, which is measured by applying a constant potential betweenthe working and reference electrodes. The signal is due toelectrochemical process involving the analyte, taking place at theelectrode surface. The potential difference is termed working potentialand is determined by cyclic voltammetry.

The ULTI immunosorbent/filter carbon modified by immobilized recombinantreplica of Hantavirus envelope was used for “sandwich immunoassay” ofHantavirus detection. ULTI carbon, containing immobilized antibodies,was deposited in the disposable sensing element as shown in bycentrifugation. The small size of carbon particles increases the rate ofimmuno-interaction at each stage. Horseradish peroxidase (HRP) attachedas a label to the anti-human IgG catalyses the oxidation of iodide intoiodine. Electrochemical reduction of iodine forms the basis fordetermination of the activity of HRP enzyme and quantification of theenzyme label. The scheme of reaction is as follows:2I⁻H₂O₂+2H⁺I₂+2H₂O  Equation 1I₂+2e−→2I⁻  Equation 2

The amount of iodine formed by reaction Equation 1, detected usingreaction Equation 2, is a measure of the activity of HRP label. Sincethe amount of antigen (analyte) determines the amount of HRP-labeledantibodies that bind to form the sandwich, amperometric measurement ofiodine formed is directly proportional to the analyte concentration. Thesandwich complex replaced by alkaline phosphatase labeledimmunoconjugate (AP) where HRP was previously used, where the hydrolysisof α-Naphthyl Phosphate to α-Naphthol is determined amperometrically.α-Naphthyl Phosphate+H₂O----AP---->α-Naphthol+HPO₄ ⁻  Equation 3

Results

The assay procedure described above was used initially to test theresponse of the system using rabbit IgG as the analyte. The rabbit IgGis immobilized on the immunosorbent and anti-rabbit IgG is used asantibody. Both peroxidase labeled and alkaline phosphatase labeledanti-rabbit IgG were used separately. FIG. 15 Tables 1 and 2 show theresponse of the system using these conjugates.

The data for both the type of the conjugates indicate the data isreproducible. But the alkaline phosphatase labeled conjugate gave highercurrent than peroxidase labeled conjugate. This implies that thereaction of α-Naphthyl Phosphate to α-Naphthol yields higher currentthan the reduction of iodine to iodide. Hence further experiments weredone using alkaline phosphatase labeled conjugate.

Mice blood samples were then tested by the same assay procedure. Therecombinant replica of Sin Nombre Virus (SNV) protein was immobilized onthe immunosorbent. Phosphatase labeled Goat anti-peromyscus leucopus IgGwas used as the conjugate. The table below gives the results obtainedfrom the device and that from genomic determination. See FIG. 15 Table3.

The next step was to evaluate the potentiostat circuit. To do this theelectrode system was connected to the potentiostat circuit and potentialwas applied on a buffer solution. Varying the resistance varied thepotential applied and the output current was measured. Similarly astandard potentiostat was taken and various potentials were applied andthe output current was noted. A graph between current and voltage wasplotted in FIG. 16. Both the results gave almost a linear dependence.

Taking a negative blood sample and diluting it to very lowconcentrations, the negative control current was obtained. Similarly apositive control was obtained by diluting positive sample to lowconcentrations. Hence if the response current is above the positivecontrol it can be said that the given sample is positive for Hantavirusantibodies. If the response is below that of the negative controlcurrent it can be said that the sample is negative. If the response liesbetween these values, the sample cannot be determined.

No false positive or false negative was obtained.

CONCLUSION

The potential of the portable amperometric immunosensor based on arecombinant nucleocapsid antigen and highly dispersed flowimmunoelectrode for fast determination of hantavirus infection in miceblood serum under field conditions has been demonstrated. The systemdesign is based on an amperometric immunosensor that employs disposablesensing elements. This approach combines the advantages of utilizationof highly dispersed immunosorbent, flow-through scheme of immunoassayand, highly sensitive electrochemical determination of enzyme label. Amain benefit of recombinant nucleocapsid antigen application inimmunoassay is that antigen preparation is easy to standardize. Sincethe principle of disposable sensing elements is involved, noregeneration of immunosorbent between measurements is required.

The potential of the portable amperometric immunosensor based on totalE. Coli, L. monocytogenes, and campylobacter C. Jejuni as antigen andhighly dispersed flow immunoelectrode for fast determination of theabove infection in blood serum, drinks, food, saliva or urine underfield conditions has been demonstrated. The system design is based on anamperometric immunosensor that employs disposable sensing elements. Thisapproach combines the advantages of utilization of highly dispersed or afilter of immunosorbent, flow-through scheme of immunoassay and, highlysensitive electrochemical determination of enzyme label. A main benefitof total E. Coli, of L. monocytogenes, and campylobacter C. Jejuniconcentration or recombinant nucleocapsid antigen application inimmunoassay is that antigen preparation is easy to standardize. Sincethe principle of disposable sensing elements is involved, noregeneration of immunosorbent between measurements.

The format of the assay and its proven applicability make it suitablefor standardized used at field located at remote sites, with minimaltechnical expertise. The handy immunosensor can be used to discrete thesamples (positive and negative) without any pretreatment of the samplesin a well-defined time interval (25 minutes). The device is extremelyuseful for studies relating to population screening of hantavirus inmice or human blood in remote areas with limited facilities, to simplifyblood collection and reduce costs without unduly sacrificing analyticalaccuracy. The device can be easily adapted for fast analysis of othermicroorganisms in biological, physiological and analytical practicesunder non-laboratory conditions and field operations such as bacteria,toxins and parasites.

1. A disposable unit for a flow through immunoassay comprising: a hollowhousing with two ends, with the first end being an opening and thesecond end being an opening for the flow; and a layer of filter orimmunosorbent having immobilized antibodies that bind to a targetanalyte in a sample solution, said layer comprising carbon paper,graphite, or ultra low temperature isotropic carbon.
 2. An assembly tobe used in a flow through immunoassay comprising: an immunosorbent layerhaving immobilized antibodies that bind to a target analyte in a samplesolution; a screen printed electrode having at least one currentcollector, said electrode is configured to form electrical contact withsaid immunosorbent layer; a holder into which a disposable unit may beinserted with an outlet capable of contacting solutions during theimmunoassay, said disposable unit is configured to permit insertion of afilter; a three electrode system; and a microcontroller.
 3. An assemblyto be used in a flow through immunoassay, comprising: a layer ofimmunosorbent having immobilized antibodies that bind to a targetanalyte in a sample solution; a screen printed electrode comprising ofat least one current collector said electrode is configured to formelectrical contact with said immunosorbent layer; a holder into which adisposable unit may inserted, with inlets which are capable ofcontacting solutions during the immunoassay, said disposable unit isconfigured to permit insertion of a filter; and; a holder with an outletwhich goes to disposal; a three electrode system; and a microcontroller.